HORMONAL-REGULATION OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR AND PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 IN CULTURED MONKEY SERTOLI CELLS

Sertoli cells play a central role in the control and maintenance of spermatogenesis. Isolated Sertoli cells of mouse and rat testes have been shown to secrete plasminogen activator (PA) and a plasminogen activator inhibitor type-1 (PAI-1) in culture. In this study, we have investigated the hormonal regulation of PA and PAI-1 activities in cultured monkey Sertoli cells. Sertoli cells (5x10(5) cells/well) isolated from infant rhesus monkey testes were preincubated at 35 degrees C for 16 h in 24-well plates precoated with poly(D-lysine) (5 mu g/cm(2)) in 0.5 mi McCoy's 5a medium containing 5% of fetal calf serum and further incubated for 48 h in 0.5 mi serum-free medium with or without various hormones or other compounds, PA as well as PAI-1 activities in the conditioned media were assayed by fibrin overlay and reverse fibrin autography techniques respectively. The Sertoli cells in vitro secreted only tissue-type PA (tPA), no detectable amount of urokinase-type PA (uPA) could be observed, Monkey Sertoli cells were also capable of secreting PAI-1, Immunocytochemical studies indicated that both tPA and PAI-1 positive staining localized in the Sertoli cells, spermatids and residual bodies of the seminiferous epithelium; Northern blot analysis further confirmed the presence of both tPA and PAI-1 mRNA in monkey Sertoli cells. Addition of follicle-stimulating hormone (FSH) or cyclic adenosine monophosphate (cAMP) derivatives or cAMP-generating agents and gonadotrophin-releasing hormone (GnRH) agonist or phorbol ester (PMA) to the cell culture significantly increased tPA activity. PAI-1 activity in the culture was also enhanced by these reagents except 8-bromo-dibutyryl-cAMP, forskolin and 3-isobutyl-1-methylxanthin (MIX) which greatly stimulated tPA activity, whereas decreased PAI-1 activity, implying that neutralization of PAI-1 activity by tile high level of tPA in the conditioned media may occur. These data suggest that increased intracellular signals which activate protein kinase A (PKA), or protein kinase C (PKC) can modulate Sertoli cell tPA and PAI-1 activities, The concomitant induction of PA and PAI-1 by the same reagents in the Sertoli cells may reflect a finely tuned regulatory mechanism in which PAI-1 could limit the excession of the proteolysis.

Inductive expression and characterization analysis of Paralichthys olivaceus pigment epithelium-derived factor in a virally infected cell line

Pigment epithelium-derived factor (PEDF) is acknowledged to be a non-inhibitory member of the serine protease inhibitor (serpin) superfamily, with antiangiogenesis, and neuroprotective and immumoregulatory function, mainly in the tissues of nervous system. Here, A PEDF gene homolog, Paralichthys olivaceus PEDF (PoPEDF), was isolated from flounder embryonic cells (FEC) treated with UV-inactivated Grass carp hemorrhage virus (GCHV) and subsequently identified as a differentially expressed gene. The full length of PoPEDF cDNA is 1803 bp with an open reading frame of 1212 bp encoding a 403-amino-acid protein. This deduced protein contains an N-terminal signal peptide, a glycosylation site, a consensus serpin motif, and a 34-mer and a 44-mer fragment, all of which are very conserved in the PEDF family. PoPEDF gene exhibits a conserved exon-intron arrangement with 8 exons and 7 introns. This conserved evolutionary relationship was further confirmed by a phylogenetic analysis, where fish PEDFs and mammalian members formed a well-supported clade. Constitutive expression of PoPEDF was widely detected in many tissues. In response to UV-inactivated GCHV or poly(I:C), PEDF mRNA was upregulated in FEC cells with time. This is the first report on the transcriptional induction of PEDF in virally infected cells. (C) 2005 Elsevier Inc. All rights reserved.

Homologous feeder cells support undifferentiated growth and pluripotency in monkey embryonic stem cells

In the present study, five homologous feeder cell lines were developed for the culture and maintenance of rhesus monkey embryonic stem cells (rESCs). Monkey ear skin fibroblasts (MESFs), monkey oviductal fibroblasts (MOFs), monkey follicular granulosa fibroblast-like (MFG) cells, monkey follicular granulosa epithelium-like (MFGE) cells, and clonally derived fibroblasts from MESF (CMESFs) were established and compared with the ability of mouse embryonic fibroblasts (MEFs) to support rESC growth. MESF, MOF, MFG, and CMESF cells, but not MFGE cells, were as good as or better than MEFs in supporting undifferentiated growth while maintaining the differentiation potential of the rESCs. In an effort to understand the unique properties of supportive feeder cells, expression levels for a number of candidate genes were examined. MOF, MESF, and MEF cells highly expressed leukemia inhibitory factor, ciliary neurotrophic factor, basic fibroblast growth factor, stem cell factor, transforming growth factor PI, bone morphogenetic protein 4, and WNT3A, whereas WNT2, WNT4, and WNT5A were downregulated, compared with MFGE cells. Additionally, all monkey feeder cell lines expressed Dkk1 and LRP6, antagonists of the WNT signaling pathway, but not WNT1, WNT8B, or Dkk2. rESCs grown on homologous feeders maintained normal karyotypes, displayed the characteristics of ESCs, including morphology, alkaline phosphatase, Oct4, the cell surface markers stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor-related antigen (TRA)-1-60, and TRA-1-81, and formed cystic embryoid bodies in vitro that included differentiated cells representing the three major germ layers. These results indicate that the four homologous feeder cell lines can be used to support the undifferentiated growth and maintenance of pluripotency in rESCs.

D77, one benzoic acid derivative, functions as a novel anti-HIV-1 inhibitor targeting the interaction between integrase and cellular LEDGF/p75

Integration of viral-DNA into host chromosome mediated by the viral protein HIV-1 integrase (IN) is an essential step in the HIV-1 life cycle. In this process, Lens epithelium-derived growth factor (LEDGF/p75) is discovered to function as a cellular co-fa

Localization and distribution of tissue type and urokinase type plasminogen activators and their inhibitors type 1 and 2 in human and rhesus monkey fetal membranes

Fetal membranes consist of 10 distinct layers including components of amnion, chorion and decidua, the latter being of maternal origin. They form mechanically integrated sheets capable of retaining amniotic fluid and play an essential role in protecting fetal growth and development in the pregnant uterus. The extracellular matrix, substrate for plasminogen activators (PAs), is an important supportive framework of the fetal membranes. :Fetal membranes from women with preterm premature rupture of membranes may differ in their protease activity compared with normal membranes. To identify the presence of PAs and their inhibitors (PAI) and their possible role in the process of fetal membrane rupture, this study in investigated the distribution and localization of both protein and mRNA for tissue (t) and urokinase (u) PA and their inhibitors type 1 (PAI-1) and type 2 (PAI-2) in amniochorion of human and rhesus monkey using conventional and. confocal immunofluorescence microscopy. In situ hybridization analysis showed that the distribution and localization of mRNAs for tPA, uPA, PAI-I and PAI-2 were similar in the fetal membranes of human and rhesus monkey; no obvious species difference was observed. Evidence of tPA mRNA was detected in amniotic epithelium, trophoblast cells and nearly all cells of the decidual layer. Strong expression of uPA mRNA was noted in the decidual cells which increased in intensity as the abscission point was approached. Weak staining in chorion laeve trophoblast was also detected. In situ hybridization experiments showed PAI-1 mRNA to be concentrated mainly in the decidual cells, some of which were interposed into the maternal-facing edge of the chorion laeve. Maximal labelling of the decidua occurred towards the zone of abscission. Weak expression of PAI-1 mRNA nas also noted in some cells of the chorion laeve. The distribution of PAI-2 mRNA in amniochorion was also concentrated in the cells of the decidual layer, maximum expression of the mRNA was in the level of abscission. No detectable amount of mRNAs for tPA, uPA, PAI-1 and PAI-2 was found in the fibroblast, reticular and spongy layers. Distribution of the proteins of tPA, uPA and PAI-1 in the fetal membranes of these two species was consistent with the distribution of their mRNA. Anti-PAI-2 immunofluorescence was found to be strongly concentrated in the amniotic epithelium, but PAI-2 mRNA was negative in this layer, suggesting that the epithelium-associated PAI-2 is not of epithelial origin. These findings suggest that a local fibrinolysis in fetal membranes generated by precisely balanced expression of PAs and their inhibitors via paracrine or autocrine mechanisms may play an essential role in fetal membrane development, maturation and in membrane rupture. Following an analysis of the distribution and synthesis of activators and inhibitors it was found that they may play a role in abscission during the third stage of labour. (C) 1998 W. B. Saunders Company Ltd.

Expression and regulation of orphan receptor TR2 mRNA in germ cells of cryptorchid testis in rat and rhesus monkey

Expression and cellular localization of orphan receptor TR2 mRNA in relation to germ cell apoptosis in cryptorchid testes of rat and rhesus monkey have been studied by using in situ hybridization and in situ 3'-end labeling of DNA fragments (TUNEL). The results show that: (i) TR2 mRNA is specifically expressed in the germ cells, mainly in the spermatocytes, round and elongated spermatids. The expression level of TR2 mRNA varies with the seminiferous cycle, (ii) In the rat cryptorchid testes on days 3 and 5 after the surgery, the germ cells began to undergo apoptosis with no evident decrease in TR2 mRNA level. On day 7.5, however, most germ cells underwent apoptosis, while the expression level of TR2 mRNA declined markedly, and TR2 mRNA was rarely expressed on day 10 thereafter. (iii) On days 15 and 20 of the cryptorchid testes of rhesus monkey, TR2 mRNA was only expressed in a few of primary spermatocytes and the mRNA was almost undetectable on days 30, 45, 60. These results suggest that TR2 mRNA probably plays an important role in spermatogenesis and germ cell apoptosis.

Effects of testosterone undecanoate as a male contraceptive candidate on rat immunological features

Testosterone undecanoate (TU) is under phase III clinical trial as a hormonal male contraceptive in China. Sex hormones can modulate the immune system. Female hormonal contraceptives may affect SIV/HIV-1 transmission. To evaluate the safety of TU and to understand whether long-term use of TU for a male contraceptive affects users' immunological features, adult male rats were treated for a 32-week TU-treated phase at the dose of 20 mg TU/kg body weight and a 24-week recovery phase. The reproductive and immunological parameters of 4-6 rats in each subgroup were examined at the stated time point. The mean sperm count and viability in the treated rats were significantly suppressed (p < 0.01). In the TU-treated group: the mean blood leukocyte and lymphocyte counts; the proliferation indexes of T cells from peripheral blood mononuclear cells (PBMC) and spleen; and, of B cells from spleen, as well as the mean counts of blood T, NK, and B cells decreased in comparison with those of control group. These decreases were not significant (p > 0.01). Similarly, the mean serum IgM, IgG, and IgA levels and complement activity in TU-treated rats were lower than those in control group (p > 0.01), and the changes in the antibody levels of the examined genital secretions were not significant (p > 0.01). The changes in the thickness of urethra epithelium, and in secretory component (SC) expression in genitals were not observed in the treated group. These results demonstrated that long-term supraphysiological TU injection did not obviously affect the examined rat immunological parameters.

Predominant expression and cellular distribution of fish Agr2 in renal collecting system

Anterior gradient 2 (Agr2) genes encode secretory proteins, and play significant roles in anterior-posterior patterning and tumor metastasis. Agr2 transcripts were shown to display quite diverse tissue distribution in different species, and little was known about the cellular localization of Agr2 proteins. In this study, we identified an Agr2 homologue from gibe[ carp (Carassius auratus gibelio), and revealed the expression patterns and cellular localization during embryogenesis and in adult tissues. The full-length cDNA of CagAgr2 is 803 nucleotides (nt) with an open reading frame of 510 nt encoding 169 amino acids. The Agr2 C-terminus matches to the class I PDZ-interacting motif, suggesting that it might be a PDZ-binding protein. During embryogenesis, CagAgr2 was found to be transcribed in the mucus-secreting hatching gland from tailbud stage and later in the pharynx region, swim bladder and pronephric duct as revealed by RT-PCR and whole mount in situ hybridization. In the adult fish, its transcription was predominantly confined to the kidney, and lower transcription levels were also found in the intestine, ovary and gills. To further localize the Agr2 protein, the anti-CagAgr2 polyclonal antibody was produced and used for immunofluorescence observation. In agreement with mRNA expression data, the Agr2 protein was localized in the pronephric duct of 3dph larvae. In adult fish, Agr2 protein expression is confined to the renal collecting system with asymmetric distribution along the apical-basolateral axis. The data provided suggestive evidence that fish Agr2 might be involved in differentiation and secretory functions of kidney epithelium. (C) 2009 Elsevier Inc. All rights reserved.

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2

Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-kappa B, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-kappa B activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but not TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1). antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and triggers innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules. (C) 2007 Elsevier Ltd. All rights reserved.

Androgen receptor targets NFKB and TSPI to suppress prostate tumor growth in vivo

The androgen role in the maintenance of prostate epithelium is subject to conflicting opinions. While androgen ablation drives the regression of normal and cancerous prostate, testosterone may cause both proliferation and apoptosis. Several investigators note decreased proliferation and stronger response to chemotherapy of the prostate cancer cells stably expressing androgen receptor (AR), however no mechanistic explanation was offered. In this paper we demonstrate in vivo anti-tumor effect of the AR on prostate cancer growth and identify its molecular mediators. We analyzed the effect of AR on the tumorigenicity of prostate cancer cells. Unexpectedly, the AR-expressing cells formed tumors in male mice at a much lower rate than the AR-negative controls. Moreover, the AR-expressing tumors showed decreased vascularity and massive apoptosis. AR expression lowered the angiogenic potential of cancer cells, by increasing secretion of an anti-angiogenic protein, thrombospondin-1. AR activation caused a decrease in RelA, a subunit of the pro-survival transcription factor NF kappa B, reduced its nuclear localization and transcriptional activity. This, in turn, diminished the expression of its anti-apoptotic targets, Bcl-2 and IL-6. Increased apoptosis within AR-expressing tumors was likely due to the NF kappa B suppression, since it was restricted to the cells lacking nuclear (active) NF kappa B. Thus we for the first time identified combined decrease of NF kappa B and increased TSP1 as molecular events underlying the AR anti-tumor activity in vivo. Our data indicate that intermittent androgen ablation is preferable to continuous withdrawal, a standard treatment for early-stage prostate cancer. (C) 2007 Wiley-Liss, Inc.

Cultured gill epithelial cells from tilapia (Oreochromis niloticus): a new in vitro assay for toxicants

A culture gill epithelium from seawater-adapted tilapia (Oreochromis niloticus) was developed for testing PAHs and dioxin-like contaminants in seawater. The epithelia consists two to three layers of epithelial cells incorporating both pavement cells and mitochondria-rich cells (MRCs). Polarity and a stable transepithelial resistance (TER) were maintained. and closely resembled those in fish gills in vivo. The tightness (integrity) of the epithelia remained unchanged upon exposure to benzo[a]pyrene (B[a]P). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3',4,4',5-pentachlorobiphenyl (PCB#126), while a concentration-dependent response of EROD activity in the epithelia was induced within 18-24 h when the apical side was exposed to these toxicants. The 24 h EC50 of EROD activity was 2.77 x 10(-7) M for PCB#126, 1.85 x 10(-7) M for B[a]P and 7.38 x 10(-10) M for TCDD. showing: that the preparation was not only sensitive to PAHs and dioxin-like compounds, but also able to produce inductive potency of AhR agonists that generally agreed with those derived from other established in vitro and in vivo systems. The results suggest, that the cultured gill epithelia from seawater-adapted tilapia may serve as a simple. rapid and cost-effective tool for assessing exposure and potential effects of toxicants in marine waters. (C) 2004 Elsevier B.V. All rights reserved.

Effects of subretinal implant materials on the viability, apoptosis and barrier function of cultured RPE cells

Background: Subretinal microphotodiode array (MPDA) is a type of visual prosthesis used for the implantation in the subretinal space of patients with progressive photoreceptor cell loss. The present study aimed to evaluate the effect of materials for MPDA on the viability, apoptosis and barrier function of cultured pig retinal pigment epithelium (RPE) cells.Methods: Primary culture of pig RPE cells was performed and 24 pig eyes were used to start RPE culture. The third passage of the cultures was plated on different materials for MPDA and MPDAs. The tetrazolium dye-reduction assay (MTT) was used to determine RPE cell viability. Flow cytometry was measured to indicate the apoptosis rates of RPE cells on different materials. RPE cells were also cultured on microporous filters, and the transepithelial resistance and permeability of the experimental molecule were measured to determine the barrier function.Results: The data from all the methods indicated no significant difference between the materials groups and the control group, and the materials tested showed good biocompatibility.Conclusions: The materials for MPDA used in the present study had no direct toxicity to the RPE cells and did not release harmful soluble factors that affected the barrier function of RPE in vitro.

非洲爪蟾早期胚胎发育中PEDF和PEDF-R的功能以及vestigial-like家族表达图式的研究

PEDF 蛋白（Pigment epithelium-derived factor）又名“色素上皮源因子”或 “色素上皮衍生因子”，为一个多功能性分泌糖蛋白，前人研究表明PEDF 蛋白 具有神经保护性、免疫调节、抑制新生血管生成以及抑制肿瘤恶化等多种功能。 PEDF-R 是PEDF 的受体， 属于PNPLA2 （ Patatin-like phopholipase domain-containing 2 family）蛋白家族的一个新成员，PEDF 蛋白与其结合后会激 活PEDF-R 的磷脂酶A2 活性。本研究中，我们描述了非洲爪蟾PEDF 和PEDF-R 基因的表达图式及其在胚胎发育中的可能功能。RT-PCR 结果显示PEDF 是非母 源性表达，而PEDF-R 则是母源性表达的。原位杂交实验表明它们均在神经系统 中特异表达，但PEDF-R 的表达区域更加广泛，在鳃弓、眼泡和耳泡中也有表达。 通过mRNA 过表达和Morpholino(MO)阻断蛋白合成等手段发现，PEDF 功能获 得和功能缺失后胚胎几乎不受影响。然而PEDF-R 过表达后胚胎向注射一侧弯 曲，TUNEL 凋亡检测实验发现这些胚胎在注射一侧发生了凋亡。这两个基因神 经表达的特异性表明它们可能在早期神经发育中有重要功能。TUNEL 结果暗示 着PEDF-R 可能是一个与凋亡信号通路相关的受体。PEDF 功能获得和缺失并未 导致胚胎明显的表型，这表明PEDF 在非洲爪蟾中可能还存在其他的受体来行使 与PEDF-R 不同功能的途径。 果蝇的vestigial 基因编码一个转录辅助因子，在果蝇中只有一个成员，即 vestigial(vg)基因。在脊椎动物中有四个vestigial 同源基因，即vestigial-like 1,2,3,4_(vgl-1,2,3,4)。Vestigial 蛋白能作为辅助因子与果蝇中的Scalloped(Sd)蛋白 或者哺乳动物中的TEF 蛋白结合成复合体，通过Sd/TEF 蛋白的TEA/ATTS 结构 域与DNA 结合，从而调节下游基因的转录。本研究中，我们克隆了非洲爪蟾 vestigial-like 家族的四个成员，并对其在爪蟾胚胎发育过程中的表达进行研究。 RT-PCR 显示vgl-2 和vgl-3 是合子型表达的，vgl-1、vgl-4 则是母源性表达。原位 杂交显示：vgl-1 主要在神经管背部、耳泡和眼泡中表达；vgl-2 则是在肌肉、第 一二鳃弓、脊索中特异表达；vgl-3 神经胚时期在后脑有强的表达信号，从神经 胚后期到尾芽期后脑部位的表达几乎消失了，而在胚胎的头部以及神经管中开始 有微弱的表达；vgl-4 的表达较广泛，在神经管、眼泡、耳泡、肌肉以及脊索中 均有表达。在爪蟾中这四个成员的表达图式各不相同，提示它们有可能与其行使 组织特异性基因调控的功能相关，上述结果将有助于对vestigial-like 家族基因在 胚胎发育中的功能研究。

非洲爪蟾早期胚胎发育中PEDF和PEDF-R的功能以及vestigial-like家族表达图式的研究

PEDF 蛋白（Pigment epithelium-derived factor）又名“色素上皮源因子”或 “色素上皮衍生因子”，为一个多功能性分泌糖蛋白，前人研究表明PEDF 蛋白 具有神经保护性、免疫调节、抑制新生血管生成以及抑制肿瘤恶化等多种功能。 PEDF-R 是PEDF 的受体， 属于PNPLA2 （ Patatin-like phopholipase domain-containing 2 family）蛋白家族的一个新成员，PEDF 蛋白与其结合后会激 活PEDF-R 的磷脂酶A2 活性。本研究中，我们描述了非洲爪蟾PEDF 和PEDF-R 基因的表达图式及其在胚胎发育中的可能功能。RT-PCR 结果显示PEDF 是非母 源性表达，而PEDF-R 则是母源性表达的。原位杂交实验表明它们均在神经系统 中特异表达，但PEDF-R 的表达区域更加广泛，在鳃弓、眼泡和耳泡中也有表达。 通过mRNA 过表达和Morpholino(MO)阻断蛋白合成等手段发现，PEDF 功能获 得和功能缺失后胚胎几乎不受影响。然而PEDF-R 过表达后胚胎向注射一侧弯 曲，TUNEL 凋亡检测实验发现这些胚胎在注射一侧发生了凋亡。这两个基因神 经表达的特异性表明它们可能在早期神经发育中有重要功能。TUNEL 结果暗示 着PEDF-R 可能是一个与凋亡信号通路相关的受体。PEDF 功能获得和缺失并未 导致胚胎明显的表型，这表明PEDF 在非洲爪蟾中可能还存在其他的受体来行使 与PEDF-R 不同功能的途径。 果蝇的vestigial 基因编码一个转录辅助因子，在果蝇中只有一个成员，即 vestigial(vg)基因。在脊椎动物中有四个vestigial 同源基因，即vestigial-like 非洲爪蟾早期胚胎发育中PEDF 和PEDF-R 的功能以及vestigial-like 家族表达图式的研究 2 1,2,3,4_(vgl-1,2,3,4)。Vestigial 蛋白能作为辅助因子与果蝇中的Scalloped(Sd)蛋白 或者哺乳动物中的TEF 蛋白结合成复合体，通过Sd/TEF 蛋白的TEA/ATTS 结构 域与DNA 结合，从而调节下游基因的转录。本研究中，我们克隆了非洲爪蟾 vestigial-like 家族的四个成员，并对其在爪蟾胚胎发育过程中的表达进行研究。 RT-PCR 显示vgl-2 和vgl-3 是合子型表达的，vgl-1、vgl-4 则是母源性表达。原位 杂交显示：vgl-1 主要在神经管背部、耳泡和眼泡中表达；vgl-2 则是在肌肉、第 一二鳃弓、脊索中特异表达；vgl-3 神经胚时期在后脑有强的表达信号，从神经 胚后期到尾芽期后脑部位的表达几乎消失了，而在胚胎的头部以及神经管中开始 有微弱的表达；vgl-4 的表达较广泛，在神经管、眼泡、耳泡、肌肉以及脊索中 均有表达。在爪蟾中这四个成员的表达图式各不相同，提示它们有可能与其行使 组织特异性基因调控的功能相关，上述结果将有助于对vestigial-like 家族基因在 胚胎发育中的功能研究。